Solid State NMR Structure/Function Studies of Amelogenin

釉原蛋白的固态核磁共振结构/功能研究

• 批准号: 8080278
• 负责人: Wendy J Shaw
• 金额: $ 37.32万
• 依托单位: BATTELLE PACIFIC NORTHWEST LABORATORIES
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-05-01 至 2014-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8080278
• 关键词: Address; Affect; Alternative Splicing; Amelogenesis Imperfecta; Amino Acids; Atomic Force Microscopy; Automobile Driving; Binding; Defect; Dental Enamel; Development; Enamel Formation; Environment; Funding; Goals; Growth; Habits; Hydroxyapatites; In Vitro; Ionic Strengths; Kinetics; Knockout Mice; Knowledge; Leucine; Liquid substance; Mechanical Stress; Membrane Proteins; Minerals; Modification; Molecular; Molecular Biology Techniques; Molecular Models; Molecular Structure; Mutate; Mutation; Nanosphere; Play; Point Mutation; Process; Proteins; Quartz; Regulation; Research; Research Design; Research Personnel; Role; Series; Solutions; Staging; Steel; Structure; Surface; Techniques; Therapeutic; Time; Tissues; Variant; Work; amelogenin; biomineralization; bone; design; dimer; enamelin; extracellular; in vivo; insight; interfacial; leucine-rich amelogenin peptide; loss of function; molecular modeling; monomer; mutant; programs; protein function; protein protein interaction; protein structure; public health relevance; self assembly; solid state nuclear magnetic resonance

DESCRIPTION (provided by applicant): The overall goal of this research is to elucidate the interfacial mechanisms of the biomineralization proteins driving the formation of enamel. Enamel is the most highly ordered biomineralization crystal and is uniquely designed to handle abrasions and mechanical stress. Enamelins, tuftelins, ameloblastins and amelogenins are proteins present during enamel formation and all have been suggested to play a critical role in enamel development. Amelogenin consists of 90% of the protein present during enamel growth, is necessary for proper enamel formation and as such, it is the primary focus of the proposed studies. Very little is understood at a mechanistic level about how amelogenin controls crystal growth. It is known that amelogenin forms into unique self assembled nanospheres which are thought to be tied to the elongated growth of enamel crystals during development. However, the organization of the nanosphere is not well defined, and the protein- hydroxyapatite interface is not understood on a molecular level. Protein structure is thought to play a key role in the function of amelogenin as a possible crystal nucleator and growth regulator, but insight into the secondary and tertiary structure of amelogenin has eluded researchers. No single technique will fully characterize the protein-protein and protein-crystal interactions controlling enamel formation mechansims, however, recent advancements in several experimental techniques present a unique opportunity to begin addressing some of these critical questions. Relating the protein-protein and protein-surface interactions to function will be the emphasis of the proposed work, particularly focusing on the loss of function as a result of mutation. Building on our previous work,these studies will utilize a suite of techniques including solution and solid state NMR, atomic force microscopy (AFM), quartz crystal microbalance (QCM), constant composition kinetics (CCK) and molecular modeling to study critical outstanding questions in the molecular mechanism of enamel formation. Using NMR, the secondary structure and the orientation of naturally occurring mutants will be determined and compared to the structure of the wildtype protein. The affect of pH, ionic strength and protein concentration will also be investigated. AFM will be used to determine the quaternary structure of the adsorbed protein. Protein-protein interactions will be determined using solution state NMR, revealing precise residues involved in nanosphere self-assembly. To provide a correlation between structure and function, QCM and CCK will be used to investigate nucleation rates, growth inhibition and crystal modification under identical conditions used in the structural studies. Correlating the structure and orientation results with differences in growth and nucleation under similar conditions will provide crucial insight into the interfacial mechanisms used by amelogenin for exquisite control of the enamel matrix. These insights are necessary for the design of theraputic solutions to deficient enamel. More generally, these studies will provide basic insight into protein/crystal interactions dominating the formation of all biominerals. PUBLIC HEALTH RELEVANCE: Enamel is the most highly mineralized tissue in the body, and produces hydroxyapatite crystals with a strength approaching that of steel. Amelogenin is a protein that is critical to the formation of this highly organized material, but how it controls enamel formation is not understood on a molecular level. Using a combination of the most advanced techniques available, we propose to elucidate the protein-protein and protein-hydroxyapatite interaction mechanisms, insights that are necessary before long-lasting therapeutics can be designed.

描述（由申请人提供）：这项研究的总体目标是阐明驱动搪瓷形成的生物矿化蛋白的界面机制。搪瓷是最有序的生物矿化晶体，其独特而设计用于处理擦伤和机械应力。搪瓷蛋白，塔夫蛋白，蛋白蛋白和蛋白酶是搪瓷形成过程中存在的蛋白质，并且都建议所有这些都在搪瓷发育中起关键作用。氨基蛋白蛋白包括牙釉质生长过程中存在的蛋白质的90％，对于适当的搪瓷形成是必要的，因此，它是拟议研究的主要重点。关于氨基蛋白素如何控制晶体生长的机械水平，几乎没有理解。众所周知，氨基蛋白蛋白形成独特的自组装纳米球，这些纳米球被认为与发育过程中搪瓷晶体的细长生长有关。但是，纳米球的组织的定义不当，并且在分子水平上尚不了解蛋白质羟基磷灰石界面。人们认为蛋白质结构在氨基蛋白酶作为可能的晶体成核和生长调节剂的功能中起关键作用，但是对酰胺蛋白的二级和三级结构的洞察力已避免了研究人员。没有任何一项技术能够充分表征控制牙釉质形成机械的蛋白质蛋白质和蛋白质 - 晶状相互作用，但是，几种实验技术的最新进步为开始解决其中一些关键问题提供了一个独特的机会。将蛋白质 - 蛋白质和蛋白质表面相互作用与功能相关的将是拟议工作的重点，尤其是由于突变而着重于功能丧失。在我们先前的工作的基础上，这些研究将利用一套溶液和固态NMR，原子力显微镜（AFM），石英晶体微量平衡（QCM），恒定组成动力学（CCK）和分子建模来研究关键的出色问题。使用NMR，将确定二级结构和天然突变体的方向，并将其与野生型蛋白的结构进行比较。还将研究pH，离子强度和蛋白质浓度的影响。 AFM将用于确定吸附蛋白的第四纪结构。蛋白质蛋白质相互作用将使用溶液状态NMR确定，揭示纳米圈自组装中涉及的精确残基。为了提供结构和功能之间的相关性，QCM和CCK将用于研究成核速率，在结构研究中使用的相同条件下的成核速率，生长抑制和晶体修饰。将结构和取向结果与在相似条件下的生长和成核的差异相关联，将为氨基蛋白用于精心控制搪瓷基质的界面机制提供至关重要的见解。这些见解对于设计治疗溶液以使牙釉质不足是必要的。更普遍地，这些研究将提供对蛋白质/晶体相互作用的基本见解，以主导所有生物矿物的形成。 公共卫生相关性：搪瓷是体内最矿化的组织，并产生羟基磷灰石晶体，其强度接近钢。氨基蛋白蛋白是一种对这种高度有组织的材料形成至关重要的蛋白质，但是它如何控制牙釉质形成在分子水平上却不理解。使用最先进的技术的组合，我们建议阐明蛋白质 - 蛋白质和蛋白质羟基磷灰石的相互作用机制，即在设计长期持久疗法之前所必需的见解。